A. Hematopoiesis
The process of blood cell formation whereby red and white blood cells are replaced through the division of cells located in the bone marrow is called hematopoiesis. For a review of hematopoiesis see Dexter and Spooncer (Ann. Rev. Cell Biol., 3:423-441 [1987]).
There are many different types of blood cells which belong to distinct cell lineages. Along each lineage, there are cells at different stages of maturation. Mature blood cells are specialized for different functions. For example, erythrocytes are involved in O.sub.2 and CO.sub.2 transport; T and B lymphocytes are involved in cell and antibody mediated immune responses, respectively; platelets are required for blood clotting; and the granulocytes and macrophages act as general scavengers and accessory cells. Granulocytes can be further divided into basophils, eosinophils, neutrophils and mast cells.
Each of the various blood cell types arises from pluripotent or totipotent stem cells which are able to undergo self-renewal or give rise to progenitor cells or Colony Forming Units (CFU) that yield a more limited array of cell types. As stem cells progressively lose their ability to self-renew, they become increasingly lineage restricted. It has been shown that stem cells can develop into multipotent cells (called "CFC-Mix" by Dexter and Spooncer, supra). Some of the CFC-Mix cells can undergo renewal whereas others lead to lineage-restricted progenitors which eventually develop into mature myeloid cells (e.g., neutrophils, megakaryocytes, macrophages, basophils and erythroid cells). Similarly, pluripotent stem cells are able to give rise to PreB and PreT lymphoid cell lineages which differentiate into mature B and T lymphocytes, respectively. Progenitors are defined by their progeny, e.g., granulocyte/macrophage colony-forming progenitor cells (GM-CFU) differentiate into neutrophils or macrophages; primitive erythroid burst-forming units (BFU-E) differentiate into erythroid colony-forming units (CFU-E) which give rise to mature erythrocytes. Similarly, the Meg-CFU, Eos-CFU and Bas-CFU progenitors are able to differentiate into megakaryocytes, eosinophils and basophils, respectively.
The number of pluripotent stem cells in the bone marrow is extremely low and has been estimated to be in the order of about one per 10,000 to one per 100,000 cells (Boggs et al., J. Clin. Inv., 70:242 [1982] and Harrison et al., PNAS, 85; 822 [1988]). Accordingly, characterization of stem cells has been difficult. Therefore, various protocols for enriching pluripotent stem cells have been developed. See, for example, Matthews et al., Cell, 65:1143-1152 [1991]; WO 94/02157; Orlic et al., Blood, 82(3):762-770 [1993]; and Visser et al., Stem Cells, 11(2):49-55 [1993].
Various lineage-specific factors have been demonstrated to control cell growth, differentiation and the functioning of hematopoietic cells. These factors or cytokines include the interleukins (e.g., IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (M-CSF), erythropoietin (Epo), lymphotoxin, steel factor (SLF), tumor necrosis factor (TNF) and gamma-interferon. These growth factors have a broad spectrum of activity, from generalized to lineage-specific roles in hematopoiesis, or a combination of both. For example, IL-3 appears to act on multipotent stem cells as well as progenitors restricted to the granulocyte/macrophage, eosinophil, megakaryocyte, erythroid or mast cell lineages. On the other hand, Epo generally acts on fairly mature erythroid progenitor cells.
B. Tyrosine Kinases
Many cytokines involved in hematopoietic development stimulate receptor protein tyrosine kinases (pTKs). For example, the c-kit pTK and its cognate ligand (KL) have been shown to play a role in hematopoiesis. Tyrosine kinases catalyze protein phosphorylation using tyrosine as a substrate for phosphorylation. Members of the tyrosine kinase family can be recognized by the presence of several conserved amino acid regions in the tyrosine kinase catalytic domain (Hanks et al., Science, 241:42-52 [1988]). Receptor protein tyrosine kinases share a similar architecture, with an intracellular catalytic portion, a hydrophobic transmembrane domain and an extracellular ligand-binding domain. The extracellular domains (ECDs), which are responsible for ligand binding and transmission of biological signals, have been shown to be composed of a number of distinct structural motifs. The intracellular domain comprises a catalytic protein tyrosine kinase. The binding of ligand to the extracellular portion is believed to promote dimerization of the pTK resulting in transphosphorylation and activation of the intracellular tyrosine kinase domain (see Schlessinger et al., Neuron, 9:383-391 [1992]).
C. Flk2/Flt3 Receptor
A murine gene encoding a pTK which is expressed in cell populations enriched for stem cells and primitive uncommitted progenitors has been identified and is called "fetal liver kinase-2" or "flk-2" by Matthews et al. in Cell, 65:1143-52 [1991]. Rosnet et al. independently identified partial cDNA sequences for the same gene, which they call "flt3", from murine and human tissues (Genomics, 9:380-385 [1991]). The full length flt3 sequence has been published by Rosnet et al. in Oncogene, 6:1641-1650 [1991]. The sequence for human flk2 is disclosed in WO 93/10136. Kuczynski et al. refer to a gene called "STK-1" which is said to be the human homologue of murine flk2/flt3 (Blood, 82(10):PA486 [1993]).
Matthews et al. isolated the flk2 cDNA from stem cell-enriched hematopoietic tissue. In order to enrich for stem cells, murine fetal liver cells were fractionated using the AA4.1 monoclonal antibody and a cocktail of antibodies raised against specific differentiation antigens, collectively called "Lin". Flk-2 was found to be expressed in AA4.sup.+, AA4.sup.+ Lin.sup.lo and AA4.sup.+ Lin.sup.br cells, but not in AA4.sup.- cells. The AA4.sup.+ Lin.sup.lo population contained all of the long-term pluripotent stem cells. The AA4.sup.+ Lin.sup.br population was depleted of pluripotent stem cells but contained multipotent progenitors. The AA4.sup.- population was devoid of primitive clonogenic cells but contained less primitive progenitors such as the CFU-E. Expression of flk2 in AA4.sup.+ Sca.sup.+ and AA4.sup.+ Sca.sup.+ Lin.sup.lo populations, which are considered to be highly enriched stem cell populations, was further demonstrated. Additional expression of flk2 in the day 14 thymus (at which stage the thymocyte population is highly enriched in primitive precursors) was studied. Flk2 mRNA was expressed in the most immature T lymphocyte population (CD4.sup.- 8.sup.- Thy-l.sup.lo /IL-2R.sup.-). Overall, the results of the experiments described in Matthews et al. indicate that flk2 is expressed in the most primitive stem/progenitor hematopoietic cells.
Poly(A).sup.+ RNA expression in fetal and adult tissues was also investigated by Matthews et al. Expression of flk2 mRNA in the fetal brain and liver as well as adult brain and bone marrow tissues was observed. Rosnet et al. similarly observed that the flt3 gene is expressed in placenta and in various adult tissues including gonads and the brain as well as hematopoietic cells (Oncogene, 6:1641-1650 [1991]). The flt3 transcript identified by Rosnet et al. was 3.7 kb long, except in the testis, where two shorter transcripts were identified.
Small et al. (USA PNAS, 91:459-463 [1994]) have shown that antisense oligonucleotides directed against the human homologue of the flk2/flt3 gene inhibit colony formation in long term bone marrow cultures, which results further indicate that flk2/flt3 may transduce growth signals in hematopoietic stem cells.
WO 94/01576 refers to a soluble form of the flk2/flt3 receptor, designated flk-2ws, encoded by a 1.9 kb DNA fragment.
Dosil et al. prepared a chimeric receptor which consisted of the extracellular ligand-binding domain of the human fms pTK and the transmembrane and tyrosine kinase domains of murine flk2/flt3. It was shown that the chimeric receptor conferred transformed properties to NIH 3T3 cells and sustained long-term proliferation of the Ba/F3 cell line (a murine IL-3-dependent hematopoietic cell line which generates B lymphocytes in vivo) in the absence of IL-3 (Mol. Cell. Biol., 13(10):6572-6585 [1993]). It was shown that flk2/flt3 interacts with the p85 subunit of PI 3'-kinase and induced tyrosine phosphorylation of PLC.gamma., GAP and Shc proteins. PI 3'-kinase, PLC.gamma., GAP and Shc proteins are intracellular substrate proteins which are known to associate with pTKs.
The flk2/flt3 receptor is structurally related to subclass III pTKs such as .alpha. and .beta. platelet-derived growth factor receptors (PDGF-R), colony-stimulating factor (CSF-1, also known as macrophage colony stimulating factor, M-CSF) receptor (c-fms) and Steel factor (also known as mast cell growth factor, stem cell factor or kit ligand) receptor (c-kit). These receptors form a subfamily of pTKs which have five immunoglobulin-like segments in their ECDs and the intracellular catalytic domains thereof are interrupted by a specific hydrophilic "interkinase" sequence of variable length. The genes of this pTK subclass appear to have major growth and/or differentiation functions in various cells, particularly in the hematopoietic system and in placental development (see Rosnet et al. in Genomics, supra). Signaling through the c-fms receptor regulates the survival, growth and differentiation of monocytes. Steel factor (SLF) which interacts with c-kit stimulates the proliferation of cells in both myeloid and lymphoid lineages and is a potent synergistic factor in combination with other cytokines (Lyman et al., Oncogene, 8:815-822 [1993]).
The flk2/flt3 pTK is mentioned by various other authors. See, for example, Orlic et al., supra; Birg et al., Blood, 80(10):2584-2593 [1992]; and Visser et al., supra.
Lyman et al. refer to the molecular cloning of the transmembrane ligand for flk2/flt3 which is shown to activate the flk2/flt3 receptor (Cell, 75:1157-1167 [1993]). The protein was found to be similar in size and structure to the cytokines, M-CSF and SLF. The flk2/flt3 ligand was shown to increase thymidine incorporation in early hematopoietic cell precursors.
In their earlier publication, Lyman et al. refer to the production of rabbit polyclonal antibodies against the interkinase domain or C-terminus of flk2/flt3 which immunoprecipitated a major band of 143 kDa and a more diffuse band of 158 kDa. A C-terminal peptide of the flt3 sequence containing the final 22 amino acids thereof was used to generate the antisera. See Lyman et al., Oncogene, 8:815-822 [1993]. Maroc et al. also refer to the production of polyclonal antibodies against the C-terminal kinase domain of flk2/flt3 for use in studying the biochemical features of this protein (see Oncogene, 8:909-918 [1993]). Polyclonal rabbit immune serum was directed against a fusion of the interkinase domain of flk2/flt3 with TrpE. However, agonist antibodies which are able to activate the flk2/flt3 receptor have heretofore not been disclosed.
D. Therapeutic Implications
Chemo- and radiation therapies cause dramatic reductions in blood cell populations in cancer patients. At least 500,000 cancer patients undergo chemotherapy and radiation therapy in the US and Europe each year and another 200,000 in Japan. Bone marrow transplantation therapy of value in aplastic anemia, primary immunodeficiency and acute leukemia (following total body irradiation) is becoming more widely practiced by the medical community. At least 15,000 Americans have bone marrow transplants each year. Other diseases can cause a reduction in entire or selected blood cell lineages. Examples of these conditions include anemia (including macrocytic and aplastic anemia); thrombocytopenia; hypoplasia; immune (autoimmune) thrombocytopenic purpura (ITP); and HIV induced ITP.
A pharmaceutical product which is able to enhance reconstitution of blood cell populations in these patients would clearly be of therapeutic benefit.
Accordingly, it is an object of the present invention to provide agonist antibodies against the flk2/flt3 receptor. The labeled antibodies can be used to detect the flk2/flt3 receptor in biological samples.
It is a further object of this invention to provide a method for enhancing the proliferation or differentiation of primitive hematopoietic cells, thus enhancing repopulation of mature blood cell lineages. This is desirable where a mammal has suffered a decrease in hematopoietic or mature blood cells as a consequence of disease, radiation or chemotherapy. This method is also useful for generating mature blood cell lineages from hematopoietic cells ex vivo.
These and other objects will be apparent to the ordinary artisan upon consideration of the specification as a whole.